研究目的
Investigating the interactions between ZnO nanomaterials and biologically relevant proteins (LDH, IL-6, IL-8) to understand confounding factors in in vitro toxicity endpoints.
研究成果
ZnO MNs cause significant reduction in LDH levels due to physisorption and potential inhibition by dissolved Zn, leading to artefacts in in vitro toxicity assays. Controls for particle interference are essential to avoid misinterpretation. The findings emphasize the need for careful assay design and validation in nanotoxicology studies.
研究不足
The study is limited to ZnO nanomaterials and specific proteins (LDH, IL-6, IL-8); results may not generalize to other MNs or assays. Experimental conditions mimic but may not fully replicate in vivo environments. pH sensors had detection limits (pH>9), potentially missing extreme values. Dissolution and adsorption mechanisms are complex and not fully elucidated.
1：Experimental Design and Method Selection:
The study followed the NANOGENOTOX batch dispersion protocol to prepare ZnO MNs dispersions. Interactions were assessed by incubating particles with proteins in complete Ham's F12 medium under simulated in vitro conditions (37°C, 5% CO2). Adsorption isotherms (Langmuir and Freundlich) were applied to quantify LDH interaction.
2：2). Adsorption isotherms (Langmuir and Freundlich) were applied to quantify LDH interaction. Sample Selection and Data Sources:
2. Sample Selection and Data Sources: Two ZnO MNs (NM-110 uncoated, NM-111 coated with triethoxycaprylsilane) were used, characterized previously. Proteins included LDH, IL-6, IL-8, and BSA. Data from three independent experiments with duplicates.
3：List of Experimental Equipment and Materials:
Equipment includes Branson Sonifier S-450D for sonication, Zetasizer Nano ZS for DLS and zeta potential, ELISA reader for absorbance, SensorDish Reader for pH and dissolution monitoring, ICP-MS for Zn quantification. Materials include Ham's F12 nutrient mixture, BSA, FBS, LDH Cytotoxicity Detection Kit, ELISA kits for IL-6 and IL-
4：Experimental Procedures and Operational Workflow:
Particles were dispersed in
5：05% BSA water via sonication. Incubated with proteins in 24-well plates for 24 hours. After incubation, samples were centrifuged, and supernatants analyzed for LDH activity and cytokine levels. Kinetics studies over time, sedimentation via DLS, dissolution via ICP-MS, and pH changes were monitored. Data Analysis Methods:
Statistical analysis using R software with ANOVA and Tukey tests. Adsorption isotherms fitted using linear regression. Thermodynamic modeling with Geochemist Workbench.
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Zetasizer Nano ZS
Nano ZS
Malvern Instruments Ltd.
Measures hydrodynamic size distribution and zeta potential of particles using dynamic light scattering.
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Branson Sonifier S-450D
S-450D
Branson Ultrasonics Corp.
Used for sonication to disperse nanomaterials in aqueous media.
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ELISA reader
Spectrophotometric microplate reader for measuring absorbance in assays.
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SensorDish Reader
SDR
PreSens Precision Sensing GmbH
Monitors temperature, pH, and dissolved oxygen in real-time during experiments.
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ICP-MS
Inductively Coupled Plasma Mass Spectrometry for quantifying zinc concentrations.
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LDH Cytotoxicity Detection Kit
Roche Diagnostics GmBH
Kit for measuring lactate dehydrogenase activity via tetrazolium salt reduction.
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ELISA kits for IL-6 and IL-8
Cat. No. 555220 and 555244
BD Biosciences
Enzyme-linked immunosorbent assay kits for quantifying interleukin concentrations.
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Pierce BCA Protein Assay kit
ThermoScientific
Assay for protein quantification using bicinchoninic acid method.
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Geochemist Workbench
v.11.0
Aqueous Solutions LLC
Software for thermodynamic chemical reaction modeling.
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